IEEE TRANSACTIONS ON
`
`PLASMA
`SCIENCE
`
`A PUBLICATION OF THE IEEE NUCLEAR AND PLASMA SCIENCES SOCIETY
`
`AUGUST 2004
`
`VOLUME 32
`
`NUMBER 4
`
`ITPSBD
`
`(ISSN 0093-3813)
`
`PART Il OF THREE PARTS
`
`SPECIAL ISSUE ON NONTHERMAL MEDICAL/BIOLOGICAL TREATMENTS USING IONIZED GASES AND
`ELECTROMAGNETIC FIELDS
`
`CHRSE BOTS igo cay i oo ee ee ee ne ee R. P. Joshi, ACG. Pakhomoy, and W. R. Rogers
`
`SPECIAL ISSUE PAPERS
`Decontamination
`Mechanisms of Bacterial Spore Deactivation Using Ambient Pressure Nonthermal Discharges... .. . J.G. Birmingham
`Bacterial Inactivation Using Low-Energy Pulsed-Electron Beam... 2.2.05 5.56 cee eee cee ewes eee
`> eal Baie PR. Chalise, M.S. Rahman, H. Ghomi, Y¥. Hayashi, M. Watanabe, A. Okino, T. Ano, M. Shoda, and BE Hona
`Plasma Treatment: of Dental Cavities: A Feasibility Stldly. .. 26. 26 seaieee cee ee esses aie ee es
`Soi a aaa telah asalalal taka aveyeeiot s eneraie R. EW. Sladek; E. Stoffels, R. Walraven, P. J. A. Tielbeek, and R. A. Pocihomes
`The Effects of UV Irradiation and Gas Plasma Treatment on Living Mammalian Cells and Bacteria: A Comparative
`ANPPPOE eon seeps tes ga ere eterna
`ee erste E. A, Sosnin, E. Stoffels, M. V. Evofeev, LE. Kieft, and $. E. Kunts
`Generation of Pulsed Electric Fields for Processing Microbes..,............+-7-F Wu, 5.-¥. Tseng, and J.-C. Hung
`Cellular and Tissue Effects
`-
`Submicrosecond Intense Pulsed Electric Field Effects on Intracellular Free Calcium: Mechanisms and Effects... ... .
`eck chiteyrcal
`loca oe eaTR YS
`a RCAZer EIS EEE OTT Ee E..S. Buescher, R. R. Smith, and K. H. Schoenbach
`Effect of Pulsed. High-Power Radiofrequency Radiation on Electroporation of Mammalian Cells .......-.5-...-..
`Py tengaie caves
`cor ple. fates eqitecrin ales . DW. Jordan, R. M. Gilgenbach, M, D. Uhler, L. H. Gates, and Y. ¥. Lau
`Characterization of the Cytotoxic Effect of High-Intensity, 10-ns Duration Electrical Pulses ........5..0.. 600004
`. AG. Pakhomoy, A. Phinney, J. Ashmore, K. Walker, Ill, J. E Katb, 8. Kono, KH. Schoenbach, and M. R. Murphy
`Strength-Duration Curve for an Electrically Excitable Tissue Extended Down to Near 1 Nanosecond........... WR.
`Rogers, J. H. Merritt, J: A. Comeaux, Jr, €.
`T. Kulinel, D. FE. Moreland. D. G, Teltschik, J. H. Lucas, and M.R. Murphy
`Nonthermal GSM Microwaves Affect Chromatin Conformation in Human Lymphocytes Similar to Heat Shock... .. .
`.
`SER Gora lias" SopeSasiel
`wt
`laces wat ae pala laretar Wein etic
`a ees R. Sarimoy, L. Malmgren, E. Markova, B. Persson, and I. ¥. Belyaev
`Localized Damageof Tissues ‘Induced by Focused'Shock Waves: .... 2.064.400 0 obec bated eae e eee eee tenes
`
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`Cove Ke lepeloue siete laraeena (a2 ayisiaisseaerlwys--« [mie lciereie Stee ree eeaceomete P. Sunka, V. Babicky, M. Clupek, J. Benes, and P. Poutkowa
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`by
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`(Contents Continued on Page 1521)
`
`IEEE
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`EXHIBIT 1005
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`EXHIBIT 1005
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`1
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`(Contents Continued from Front Cover)
`
`Mode- and Cell-Type DependentCalcium Responses Induced by Electrical Stimulus. ... 0.000.006. 0ee ee eee
`Sacer eee TaN el cette Sete Ua Cn IU eed IAL ean go ete UM toesee 2 1A. Titushkin, V. 8. Rao, and M. R. Cho
`Nanosecond Electroperturbation—Mammalian Cell Sensitivity and Bacterial Spore Resistance. ..... 0.000.000.0005
`sp Mastin hte orien Saeacns MO abiicecte, aT soesPT. Vernier, M. M.S. Thu, L. Marcu, C. M. Craft, and M. A, Gundersen
`Experimental Studies on Killing and Inhibiting Effects of Steep Pulsed Electric Field (SPEF) to Target Cancer Cell and
`Sali Parse eae ac ceteeOOR w e
`ae OR A Ae C. Yao, C. Sun, Y. Mi, L. Xiong, and 8. Wang
`Whole Organism Effects
`Millimeter-Wave-Induced Hypoalgesia in Mice: Dependence on Type of Experimental Pain... ...........0000005
`Fs acltiliceladnat ele alert Biel he A. Radzievsky, O. Gordiienko, A, Cowan,§. 1. Alekseev, and M. C. Ziskin
`Rat Electrocardiogram During Acute Exposure to Synchronized Bursts of Ultra-Wideband Pulses... 0.0.00... 040006
`Tavera eae nena oatenate a mcteierara soteininpit eis ae hr tcteeal stat heinle Metrara ees ie enc R. L. Seaman and J. R, Jauchem
`Destruction of Cutaneous Melanoma With Millimeter Wave Hyperthermia in Mice 2.0... 000000000.00.0 0.000008
`sags I. Szabo, 8. 1. Alekseev, G. Acs, A. A, Radzievsky, M. K. Logani, V. R. Makar, O. R. Gordiienko, and M. C. Ziskin
`Complex Therapeutical Effect of lonized Air: Stimulation of the Immune System and Decreasein Excessive Serotonin.
`50s bs a Link between the Iwo: Coumeiparts..s cose ceva ate sises et we die eisive tebe yin elas see als cine be sins
`V. P. Tikhonov, A. A. Temnov, V. A. Kushnir, T. V. Sirota, E.G. Litvinova, M. V. Zakharchenko, and M. N. Kondrashova
`Modeling
`A Novel Waveguide-Based Radio Frequency/Microwave Exposure System for Studying Nonthermal Effects on
`Neurotransmitter Release—Finite-Difference Time-Domain Modeling. ... 2... 6.66.6 eet ete eee eee
`i lenaiy als breil ale tole omelet eee et ataiera re’
`x hieiooe ls aeaca aaa eet oet T. Hagan, I. Chatterjee, D. McPherson, and G, L. Craviso
`Modeling Studies of Cell Response to Ultrashort, High-Intensity Electric Fields—Implications for Intracellular
`Minipulatian 5 aoa ese eithae areas Weare ara ee eter noneesel R. P. Joshi, Q@. Hu, and K. H, Schoenbach
`Modeling Electrode-Based Simalidion of Muscle and Nerve by Ultrashort Electric Pulses... 0000000000000 00 000.
`eee) OE PRL MUL VIL lee, ie Avr Lae) CS se vre care R. P. Joshi, F. Chen, and W. R. Rogers
`Transport Lattice Approach to Describing Cell Electroporation: Use of a Local Asymptotic Model ..............
`FTE ye tops
`at RT SO Te, Ng et Ad Ee D. A. Stewart, Jr, T. R. Gowrishankar, and J. C. Weciver
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`IEEE TRANSACTIONS ON PLASMA SCIENCE
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`

`IEEE TRANSACTIONS ON PLASMA SCIENCE, VOL. 32, NO. 4, AUGUST 2004
`
`The Effects of UV Irradiation and Gas Plasma
`_ Treatment on Living Mammalian Cells and Bacteria:
`A Comparative Approach
`
`Edward A. Sosnin, Eva Stoffels, Michael V. Erofeev, Ingrid E. Kieft, and Sergey E. Kunts
`
`Abstract—Living mammalian cells and bacteria were exposed
`to irradiation from narrow-band UV lamps and treated with
`a nonthermal gas plasma (plasma needle). The model systems
`were: Chinese Hamster Ovary (CHO-K1) cells (fibroblasts) and
`Escherichia Coli bacteria. UV irradiation can lead to cell death
`(necrosis) in fibroblasts, but the doses that cause such damage are
`much higher than those needed to destroy Escherichia Coli, The
`usage of UV radiation in combination with active oxygen radicals
`lowers the UV dose sufficient to kill the cells, However, in any case
`the fibroblasts seem to be fairly resistant to UV radiation and/or
`radicals. Possibly, the lamps may be used for decontamination of
`infected wounds. The most important active species in an atmo-
`spheric plasma are the radicals; the role of UV is less pronounced.
`Treatment of CHO-K1cells with the plasma needle can lead to cell
`necrosis under extreme conditions, but moderate doses cause only
`a temporary interruption of cell adhesion. Plasma needle may be
`used for fine tissuetreatment (c.g., controlled cell removal without
`inflammation) and also for bacterial decontamination.
`Index Terms—Bacterial decontamination,
`fibroblast cells,
`plasma treatment, UV radiation.
`
`I,
`
`INTRODUCTION
`
`species—radicals). However, Soloshenkoet al. [3] showed that
`also the 160-220-nm UV radiation plays an importantrole in
`low-pressure plasma sterilization,
`Nowadays, a general trend has arisen both in material pro-
`cessing and biotechnology: vacuum reactors are often being re-
`placed by atmospheric plasma sources. There are already var-
`ious principles and designs, which deliver plasmas operating at
`temperatures not higher than several tens of degrees. Examples
`include atmospheric jets [5], dielectric barrier discharges [6],
`and microplasmas [7]. These new developments allow treatment
`to materials, which can withstand neither high temperature nor
`low pressure (e.g., biological tissues). Anti-bacterial properties
`of atmospheric nonthermal plasmasare already well established
`[4]. [5]. The inactivation of microorganisms proceeds faster than
`at low pressures; it is ascribed to radical interactions with the
`membrane, while the role of UV is less important [4].
`In parallel to plasma sterilization, treatment with UV radia-
`tion alone is a subject of extensive investigations [8]-|14]. EF
`fective bacterial inactivation was demonstrated for many kinds
`of bacteria and viruses; various sources were used and the wave-
`
`ONTHERMALplasmas operate at room orslightly ele-
`vated temperatures. Treatment with such plasmas has been
`demonstrated to be a powerful method of inactivating microor-
`ganisms [1]-|5]. Plasma treatment has many advantages in com-
`parison with other bacterial inactivation methods, such as dry
`heat or hot steam sterilization, irradiation by UV/gamma rays
`and other techniques [1]. Plasma decontamination is usually
`fast, efficient, and safe in terms of thermal, chemical, or irra-
`diation damage.
`Nonthermal plasmas are easiest to generate under reduced
`pressures (less than | mbar) in specially designed vacuum reac-
`tors, In the last decennia, these plasmas found a broad spectrum
`of applications in material technology. The idea of employing
`low-pressure plasmas for bacterial inactivation was introduced
`long ago [2]. Primarily, it was assumed that the key agent in
`plasma sterilization is the chemistry (highly reactive unstable
`
`Manuscript received September 26, 2003; revised April 15, 2004. This work
`was supported by the Netherlands Organization for Scientific Research (NWO),
`E. A. Sosnin and M, V. Erofeewv are with the Optical Radiation Laboratory,
`High Current Electronics Institute. SB RAS, Tomsk 634055, Russia (e-mail:
`badik@loi-heei.tsc.ru),
`E. Stoffels and 1. E. Kieft are with the Faculty of Biomedical Engineering,
`Eindhoven University of Technology, Eindhoven, The Netherlands (e-mail:
`e.stoffels.adamowicz @ tue.nl).
`5S. E. Kunts, deceased, was with the Optical Radiation Laboratory, High Cur-
`rent Electronics Institute, SB RAS, Tomsk 634055, Russia,
`Digital Object Identifier 10.1 109/TPS.2004,833401
`
`length dependence was determined | 12]-{ 14]. Furthermore, the
`synergy of active oxygen radicals and UV was studied [15]. For
`this purpose, photosensitizerslike H2O» were used. This partic-
`ular molecule produces OH, peroxy. and hydroperoxy radicals
`upon irradiation, It was shown that the joint action of radicals
`and photons facilitates bacterial inactivation.
`In the mentioned studies, UV and plasmatreatment was per-
`formed ex vive. One is reluctant to apply these media to living
`tissues because ofthe risk of cellular damage by UV [16] and
`radicals [17]. However, we have noticed that the lethal doses for
`bacteria are surprisingly low. Therefore, in this paper, we inves-
`tigate the possibility of in vive treatment, selective bacterial in-
`activation without harming the cells. For this purpose, one has
`to establish what doses of UV or plasma exposure can be ap-
`plied without extensive tissue damage. We study the reaction of
`living mammalian cells to UV radiation, using narrow-band UV
`lamps (excilamps) and Chinese Hamster Ovary (CHO-K 1) cells
`in culture as a model system, These cells are fibroblasts, a basal
`cell type, which is involved in many processes,e.g., wound re-
`pair. We compare the effects of UV radiation on cells with data
`on UV bacterial deactivation. Furthermore, the case when irra-
`diated cells are immersed in a hydrogen peroxide solution (a
`photosensitizer) is considered.
`Finally, we present somefacts on in vive treatment by means
`of a novel nonthermal plasma source (the plasma needle [7]). A
`fundamental study has been undertaken to identify all possible
`0093-38 13/04520,00 © 2004 TEBE
`
`4
`
`

`

`SOSNIN et al: EFFECTS OF UV IRRADIATION AND GAS PLASMA TREATMENT ON LIVING MAMMALIAN CELLS
`
`1545
`
`
`
`Fig. |. CHO-K1 cells in culture, used for UV and plasma treatment. Untreated
`sample. Cells are about 30 jam long.
`
`responsesofliving objects exposed to the plasma. This may lead
`to developmentof new techniques,like disinfection oftissues or
`removal of cells without inflammatory response, and on a longer
`time scale to new methods in the health care.
`
`Il. EXPERIMENTAL SETUP
`
`A, Cell Culture Preparation
`CHO-KI1 cells were cultured in flasks containing Ham's
`F-12 medium with stable L-glutamin (Bio Whittaker, Europe)
`containing 9.0% of Fetal Bovine Serum [(FBS), Biochrom,
`AG] and 0.45% of Gentamycin (10 mg/ml, Biochrom AG). The
`cells were stored in an incubator at 37 °C with 5% COs. To
`prepare samples for UV treatment, the cells were trypsinized
`(0.05% Trypsin/0.02% EDTA solution in PBS, Biochrom AG)
`and transferred into Petri dishes. For plasma treatment, the
`same procedure was followed, but the cells were transferred
`onto sterilized object glasses (26 x 10 x 1 mm, adapted to fit
`into the plasma chamber) and placed in multiwell dishes, The
`cells were incubated for 2 or 3 days. or until nearly confluent
`(see Fig. 1).
`Just before treatment, the medium was removed and the sam-
`ples were rinsed twice with phosphate buffered saline (PBS).
`During irradiation or plasma exposure the cells were covered
`with a thin film of PBS (typically 0.3 mm) in order to prevent
`them from drying.
`
`B. Bacterial Sample Preparation
`A pure culture of Escherichia Coli, belonging to the main
`species of the enterobacteria group, was used. E. Coli is one of
`the most resistant species within enterobacteria group, so it is
`frequently used in a study of UV disinfection and sanitation,
`performed at the Scientific Research Institute of Balneology
`(Tomsk, Russia). The £. Coli was supplied by the American Na-
`tional Academy, and had a series number K12 ATCC 25 922.
`The culture of £. Coli was supported on beef-extract agar
`(BEA) and kept at the temperature of 4 °C. The optimal con-
`centration of microbial dredge was found based on the method
`of multiple dilutions,
`
`C. Ultraviolet Radiation Sources
`
`Irradiation was performed using two low-pressure capacitive
`discharge UV lamps developed at Laboratory of Optical Radia-
`tion, High Current Electronics Institute, Siberian Branch of the
`Russian Academy of Science |18}-(22]. These lamps are con-
`fined in quartz tubes (38-mm inner diameter and 300-mm-long
`silica tube with 80% transparency at A = 200 nm). The first
`of these lamps is a new excimer lamp, filled with Xe and Bry
`
`mixture (Type XeBr_LERA_5); the active radiating species is
`XeBr*. Excimer lamps (or so-called excilamps) are a subclass
`of electric discharge lamps emitting in UV or vacuum UV spec-
`tral ranges. Excilamps provide UV irradiance up to 10 mW/em?
`and their efficiency is typically 7%-30%. The main feature
`which distinguishes excilamps from other UV sources is that
`their spectra consist of narrow bands (the band halfwidth rarely
`exceeds 5-8 nm). The B—X band emission adds to as much
`as 80%~-90% ofthe total radiation of excilamp. By varying the
`compositionof the working mixture it is possible to select a
`required wavelength. Therefore, excilamps are suited to study
`wavelength-specific effects of UV radiation on various objects,
`including microorganisms. The XeBr-excilamp employed in
`our experiments produces a spectrum with an emission peak
`at \ ~ 282 nm and weak D — X and C — A bands. As
`confirmed in our previous experiments [14], the XeBr-lamp is
`mostefficient in sterilization. This is because the inactivation
`of bacteria is mainly related to the DNA/RNA damage,and the
`maximum absorption of DNA and RNA occurs at the photon
`wavelength between 240 and 300 nm,
`The second light source is a low-pressure lamp (Type
`1_LERA_5)filled with iodine vapor [19]. The lodine lamp
`produces a spectrum with a narrowpeak of I* emission at
`A ~ 206 nm.
`
`D. Low-Temperature Plasma
`A small-size nonthermal plasma(plasma needle)has been de-
`veloped for the purpose of studying plasma interactions with
`living species [7]. A plasma needle is an atmospheric glow ini-
`tiated under helium atmosphere by applying radio-frequency
`(about 10 MHz)electric voltage to a sharpmetal pin. The plasma
`operates under gentle conditions; the voltage needed to sustain
`the discharge is low (200-400 V peak-to-peak), and the electric
`power consumption is only 10-300 mW.The size of the glow is
`01-1 mm. It has been previously established that the gas tem-
`perature in the plasma is at most a few degrees above the room
`temperature. Also, energy fluxesfrom the plasma have been de-
`termined for different operation parameters, using a calibrated
`probe. The total energy flux (radiation, radical. and ion contri-
`bution) ranges from.0 to 2 W/em*. The plasma can be applied to
`heat-sensitive materials or humanskin(see Fig. 3) without any
`thermal damage or pain sensation. From spectral measurements,
`it follows that this plasma is a poor UV source. Most of the
`emission (helium lines, nitrogen, and oxygen bands) lies within
`the visible range. Some No bands are detected between 300and
`400 nm, no emission can be found below 300 nm (see typical
`spectra in Fig. 4). Possibly, the plasma could emit radiation in
`the vacuum UV range (< 100 nm,originating from the helium
`transitions), which we cannot detect using the present equip-
`ment. However, these short-wavelength photons cannot propa-
`gate outside the plasma;they are efficiently cut off by air and
`water (absorption coefficient in water is 10° m~! at 100 nm).
`Theexperimental arrangementfor cell treatmentis shown in
`Fig. 5. The chamberwas filled with helium at the flow rate of
`2 l/min.Flat samples were placed on the bottom ofthe chamber,
`where an externally manipulated stage is installed. Thedistance
`between the sample surface and the needle tip was adjusted by
`means of another manipulator,
`
`5
`
`

`

`IEEE TRANSACTIONS ON PLASMA SCIENCE, VOL 32. NO. 4, AUGUST 2004
`
`o00
`
`200
`
`
`
`
`XeBr*(C-A)
`
`
`400
`
`450
`
`$00
`
`350
`
`A, nm
`
`200
`
`260
`
`300
`
`Fig. 2.
`
`Emission spectra (range 200-500 nm) from two different UV light sources: (1) iodine lamp and (2) XeBr-excilamp.
`
`He
`
`Fig. 5, Experimental arrangementfor cell treatment with the plasma needle,
`rectangular metal/plastic box filled with helium. (a) Manipulators to move the
`sample and to adjust the needle—sample distance, (b) needle, (c) sample, and
`(d) thermocouple to control the temperature.
`
`UV treatment of E. Coli bacteria was performed in Tomsk;
`Petri dishes with £. Coli cultures were irradiated from several
`seconds to several minutes at a 5-cmdistance and at different
`levels of UV irradiance (up to 10 mW/cem*). Each exposure
`wasrepeated fourto five times. For control, several dishes were
`left not irradiated. The number of survivors was determined
`by the method of bacterial
`inoculation in dense media (the
`Koch method of bacterial inoculation in agarized media in Petri
`dishes), The inoculations were cultivated at the temperature of
`20 °C-23 °C. After 72 h, the number of surviving microorgan-
`isms was determined.
`During plasma treatment, the object glasses containing the
`CHO-K1 cells were placed on the moving stage at the bottom
`of the chamber. A plasma needle was brought to a 1.5-mm dis-
`tance to the sample: visually. the glow was just touching the
`surface of the PBS solution, Typical treatment time was | min,
`during which the sample was moved by the manipulator over
`a typical distance of | cm. This produces a typical “track” of
`
`(a)
`
`(b)
`
`Plasma needle: plasma is created at the tip of a sharp metal needle.
`Fig. 3.
`(a) Treatmentof cells in culture placed in a Petri dish (for precise positioning
`another setup was used, see Fig. 5). (b) Glow spreads over the surface ofthe
`tissue,
`
`80000
`
`600
`500
`700
`wavelength (nm)
`
`«800-900
`
`0
`300
`
`a
`400
`
`Typical spectra of the plasma:in pure helium (gray line, given an offset
`Fig.4.
`of 10.000 counts) and ina mixture of 90% belium and 10% nitrogen (black line).
`Latter case, atomic helium lines are suppressed and some Ny bands emerge
`below 400 nm.
`
`E, Sample Treatment
`For UV treatment, Petri dishes were placed at a 5.5-em dis-
`tance from the UV lamp. At this distance, the heating effect of
`substrate was minimized, while the irradiance could be suffi-
`ciently high. The uniformity in the radiation dose over the sur-
`face of the sample was about 10%. UV radiation was dosed by
`varying the treatment time between 2 and 5 min, while the lamp
`irradiance was kept constant at 4 mW/cm?*for the XeBr lamp
`and 3 mW/em/? for the I-lamp. All experiments were repeated
`at least three times.
`
`6
`
`

`

`SOSNIN et al: EFFECTS OF UV IRRADIATION AND GAS PLASMA TREATMENT ON LIVING MAMMALIAN CELLS
`
`IM
`
`XeBr-excilamp irradiation
`
`Esch.
`
`coli,% 0
`10 ess=ouo
`
`Fig. 6. CHO-K1 sample irradiated by the iodine lamp at 0.6 J/em?. Most of
`the cells are necrotic.
`
`
`
`
`—e— |-lamp
`—+— XeBrlamp
`
`oO
`
`0.0 01
`05 06 O7 O8
`09
`10
`02
`0.3
`0.4
`UV dose, Vom*
`
`Survival curve of CHO-K1 after treatment with UVB (@) and UVC
`Fig. 7.
`(*) radiation. Cells were irradiated at 37 °C.
`
`5
`
`10
`
`20
`15
`Fluence, mi/cmn*
`Survival curve of Escherichia coli
`8.
`Fig.
`(XeBr-excilamp).
`100
`
`26
`
`30
`
`35
`
`irradiated with UVB
`
`KrClexcilamp irradiation
`
`aWw
`0,01
`
`16-3
`
`0
`
`50
`
`100
`
`150
`
`j
`
`200
`
`plasma-treated cells, which can be easily recognized under the
`microscope.Individual cells on this track were irradiated for |
`to 10's,
`
`Fluence, mu/em*
`Survival curve of Escherichia coli after
`9.
`Fig.
`KrCl-excilamp (222 nm).
`
`irradiation with a
`
`F. Observation
`
`After treatment, the samples were observed undera light mi-
`croscope. Trypan blue (0.5% in physiological saline, Biochrom)
`was applied in order to distinguish between dead and living
`cells. Trypan blue penetrates only the cells with membrane
`leakage (dead cells) and colors the whole cytoplasm blue:
`the living cells remain colorless. Dead and living cells were
`counted and the fraction of survivors was determined. The
`surviving fraction (SF) is defined by N/Np » 100%, where No
`is the initial number of cells and N the number of surviving
`cells. Mean values were calculated from numbers found in five
`different places of a Petri dish,
`In some cases (mainly after plasmatreatment), no trypan blue
`was applied, but the samples were incubated for several hours
`in order to study the long-term post-treatment phenomena.
`
`IIL. RESULTS AND DIsCUSSION
`
`As expected, high UV doses result in cell damage and death
`by necrosis (rupture of cell membranes), It can be seen in Fig. 6,
`where a sample treated with the iodine lamp at 0.6 J/em* and
`subsequently stained with trypan blue contains a mixture of
`dead and surviving cells. The dependence of the fraction of SF
`onthe irradiation dose is displayed in Fig. 7. The most striking
`feature is the presence of a threshold. The process of inactivation
`begins only after some critical dose of radiation, which is about
`0.5 J/em® for the iodine and 0.7 J/em? for the XeBr lamp.All
`cells within the irradiated area are dead when the dose exceeds
`
`0.7 Siem? for iodine and | J/cm? for XeBr lamp. The UV dose
`(threshold and lethal dose) is lower for UVC (I-lamp) than for
`UVB (XeBr lamp). This can be easily understood because ab-
`sorption of UV radiation by lipids in cell membranesis highest
`for 4 < 230 nm [23] and the irradiated powerof the iodine lamp
`lies mainly in this range (Fig. 2).
`The presence of the threshold dose for cell deactivation
`(Fig. 7) makes these results significantly different from the ones
`obtained for deactivation of Escherichia Coli and other bacteria,
`widely reported in the literature [10}-[15]. For comparison,
`we include typical bacterial survival curves, resulting from
`the irradiation of E. Coli with XeBr and KrCl (A = 222 nm)
`excilamps (both developed in the High Current Electronics
`Institute, Tomsk), These are given in Figs. 8 and 9, respectively.
`First of all, one can immediately see that UV doses needed
`to deactivate E. Coli are much lower than the ones that cause
`necrosis in fibroblasts. Besides, already very low UV doses
`reduce the bacterial population by two decades. At higher
`doses, the killing rate is somewhat tempered, and the survival
`curves display exponential tails. Decontamination to 0.01% of
`the original population is achieved by UV doses, which are still
`too low to affect fibroblasts (at least not on the short term, or
`on a typical time scale of 2 days during which CHO-K1 cells
`can be maintained in the incubator). This shows that fibroblasts
`are extremely resistant to UV. Irradiation with UV lamps may
`become a method of selective bacterial decontamination of
`wounds without killing the body cells that strive to repair the
`wound.
`
`7
`
`

`

`100
`
`SF,% 40
`
`—s— |-larnp
`
`00 003 O10
`
`0,30
`
`0,28
`020
`015
`UV dose, icm*
`Survival curve of CHO-K1 after irradiation with the iodine lamp in
`Fig. 10.
`presence of 3% HzO, aqueous solution, Cells were irradiated at 37 °C,
`
`0,35
`
`0.40
`
`Note that the survival curve of fibroblasts (Fig. 7) does not
`have an exponential tail. This is most likely because the cell
`counting after irradiation wasdone after two-step flushing with
`PBSsolution, In this way, small amounts ofalive cells could
`be flushed away. Besides, wecould not apply UV doses much
`higher than I J/em?,because ofcell desiccationdueto a too long
`treatment time or heat damage at a too short distance from the
`lamp.
`In order to simulate the conditions characteristic for plasma
`treatment,
`it
`is necessary to supply active radicals simulta-
`neously with UV irradiation. This can be performed using
`a photosensitizer, such as H»Oo, which produces reactive
`oxygen species upon UY irradiation, Possibly, in the current
`experiment, the conditions were similar to those described
`by Soloshenko er al. [3], whoperformed effective bacterial
`inactivation in a low-pressure plasma. However,it is difficult to
`compare the exact UV doses, because a low-pressure plasma
`emits also photons with a shorter wavelength (<200 nm).
`These photonsare normally lost by absorption in air at ambient
`pressure, but they can reach the surface when the sterilized
`object is surrounded by plasma under low-pressureconditions,
`We used radiation with A = 206 nm (I-lamp), which is not
`efficiently absorbed in air or water. A 3% aqueous H2OQ2 so-
`lution was applied to the CHO-K1 culture just before irradia-
`tion; the layer of fluid was about |-mm thick. The H»Oz solu-
`tion alone (without lampirradiation) also has some toxic effects
`on cells, because small amounts of reactive oxygen species are
`released by decomposition of H2O2 by ambientlight. However,
`the cells could survive in this environmentfor several minutes.
`The UV lampin Fig. 10 was applied to cell/H202 samples for
`up to 5 min. The last points, corresponding to long treatment
`times, may be influenced by natural toxicity of peroxide solu-
`tion. However,when the UV was applied, we observeda major
`increase ofthe killing rate, even at short treatmenttimes. Fig. 10
`demonstrates that the usageofH2O. together with UVC lowers
`the lethalradiation dose by a factor of two (compare with Fig. 7).
`Moreover, there is no threshold in the inactivation curve; the
`fraction of dead cells increases approximately linearly with in-
`creasing UV dose.
`Similar studies of theeffect of 1% H2O» incombination with
`254-nm radiation on bacteria were described by other authors
`
`IEEE TRANSACTIONS ON PLASMA SCIENCE, VOL. 32, NO. 4, AUGUST 2004
`
`[15]. They reported an efficient reduction of bacterial popula-
`tion; the UV doses neededto inactivate 99.99% of microorgan-
`isms were in some cases even ten times lower than in absence
`of a photosensitizer.
`From the above results, it can be concluded that the effect of
`UV (alone or in combination with active radicals) on fibroblasts
`is by far not so drastic as on bacteria. The rate of killing cells
`is much lower than that for E. Coli (compare linear scales in
`Figs. 7 and 10 with logarithmic scalesin